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Acute cytotoxicity in in vitro culture models is linked to intrinsic cell type susceptibility, compound dosage, and exposure period. Conventional, single-parameter, membrane integrity or cell-health assays may reveal changes in viability or overall cell number, but rarely provide valuable insight into the cytotoxic mechanism by which cells are eliminated. This mechanistic information can be critical for proactively prioritizing lead compounds for medicinal development efforts. Failure to consider multi-parametric data can lead to an incomplete decision process and potentially wasted downstream efforts and resources. We have developed a sequential addition, homogeneous, same well assay method that measures cellular viability and caspase activity as biomarker surrogates for defining cytotoxic mechanism. The resulting signals are fully compatible because the reporting molecules are spectrally distinct (fluorescence and luminescence), and are sufficiently sensitive to be miniaturized into high density plate formats. Herein we describe how the multiplex can be employed to generate distinct profiles consistent with phenotypic outcomes using a variety of cell models and pharmacologically active compounds.

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